Vitamin B6 Biosynthesis by the Malaria Parasite Plasmodium falciparum: BIOCHEMICAL AND STRUCTURAL INSIGHTS

Martin Gengenbacher,Teresa B Fitzpatrick,Thomas Raschle,Karlheinz Flicker,Irmgard Sinning,Sylke Müller,Peter Macheroux,Ivo Tews,Barbara Kappes

doi:10.1074/jbc.m508696200

Abstract

Vitamin B6 is one of nature's most versatile cofactors. Most organisms synthesize vitamin B6 via a recently discovered pathway employing the proteins Pdx1 and Pdx2. Here we present an in-depth characterization of the respective orthologs from the malaria parasite, Plasmodium falciparum. Expression profiling of Pdx1 and -2 shows that blood-stage parasites indeed possess a functional vitamin B6 de novo biosynthesis. Recombinant Pdx1 and Pdx2 form a complex that functions as a glutamine amidotransferase with Pdx2 as the glutaminase and Pdx1 as pyridoxal-5 '-phosphate synthase domain. Complex formation is required for catalytic activity of either domain. Pdx1 forms a chimeric bi-enzyme with the bacterial YaaE, a Pdx2 ortholog, both in vivo and in vitro, although this chimera does not attain full catalytic activity, emphasizing that species-specific structural features govern the interaction between the protein partners of the PLP synthase complexes in different organisms. To gain insight into the activation mechanism of the parasite bi-enzyme complex, the three-dimensional structure of Pdx2 was determined at 1.62 A. The obstruction of the oxyanion hole indicates that Pdx2 is in a resting state and that activation occurs upon Pdx1-Pdx2 complex formation.

Highlights

Plasmodium falciparum is the causative agent of severe malaria
Pdx1 and -2 Are Expressed in P. falciparum Blood Stages and Reside in the Parasite Cytosol—To assess that Pdx1 and Pdx2 are expressed during the erythrocytic life stages of P. falciparum, stage-specific parasite lysates were analyzed by Western blotting using antibodies generated against both recombinant proteins
The plasmodial pyridoxal 5Ј-phosphate (PLP) synthase is a typical class I glutamine amidotransferase (GATase) consisting of the glutaminase subunit, Pdx2, that produces ammonia from glutamine and the synthase subunit, Pdx1, which most likely tunnels the reactive intermediate to the second active site where PLP is formed from ammonia, a pentose, and a triose

Summary

Introduction

Plasmodium falciparum is the causative agent of severe malaria. Each year up to two million human deaths and enormous economic losses are attributed to this parasite. Vitamin B6 is renowned in the medical field as being involved in more bodily functions than any other single nutrient It is required for the maintenance of physical as well as mental health. The metabolically active form is pyridoxal 5Ј-phosphate (PLP), an essential co-enzyme in numerous pathways such as amino acid metabolism and the biosynthesis of antibiotic compounds. Analyses of a number of available genomes has demonstrated that most organisms, including all archaea, fungi, plants, and protozoa and most eubacteria use a class I glutamine amidotransferase (GATase) composed of two domains, a glutaminase and its associated acceptor/ synthase domain to generate vitamin B6 (8 –13). The GATase involved in vitamin B6 biosynthesis is a bi-enzyme complex consisting of Pdx, the acceptor/synthase, and Pdx, the glutaminase domain [16, 17]. In view of the absence of vitamin B6 biosynthesis in the mammalian host, this study is aimed to obtain further insights into the suitability of this metabolic pathway for the design of new antimalarials

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